Altering Fate Determination of Human Bone Marrow-Derived Mesenchymal Stem Cells through Environmental and Transcriptional Cues

Boyette, Lisa, Physiology - Graduate School of Arts and Sciences, University of Virginia
Tuan, Rocky, Orthopaedic Surgery, University of Pittsburgh

Preservation of adult stem cells pools is critical for maintaining tissue homeostasis into old age. Exhaustion of adult stem cell pools as a result of deranged metabolic signaling and premature senescence as a response to oncogenic insults to the somatic genome both contribute to tissue degeneration with age. Both progeria, an extreme example of early-onset aging, and heritable longevity have provided avenues to study regulation of the aging program and its impact on adult stem cell compartments.
Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multi-lineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor (VEGF) secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was only detected in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were up-regulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in 3-dimensional (3D) pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. While hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high quality differentiated tissue.
Media formulations for MSC culture and differentiation vary between labs; two notable variables addressed inconsistently in the field are ascorbate supplementation and glucose concentration in MSC expansion medium. Glucose and ascorbate effects are coupled to the Hif1α pathway. Therefore, we examined the effects of ascorbate supplementation and low versus high glucose medium in the settings of hypoxia and normoxia on colony formation, colony morphology, proliferation, VEGF secretion, and senescence of MSCs. We found that there is no one medium formulation that maximizes MSC performance for all of these assays. Depending on the desired outcome of the culture, the medium and other culture conditions can be adjusted to optimize for a particular aspect of MSC biology, such as colony formation when clonal selection is the desired end application or delayed senescence if MSCs are to be expanded extensively.
Not only do MSCs not retain their proliferative and multi-lineage differentiative capabilities after prolonged ex vivo propagation, there is also evidence to suggest MSCs in aging humans exhibit functional decline. Modifying MSCs to resist aging during ex vivo propagation would yield a favorable cell source for regenerative medicine applications. We developed and tested novel methods for partial transcriptional reprogramming of bulk MSC populations and investigated whether this technique is effective for enhancing function in MSCs subjected to in vitro aging.

PHD (Doctor of Philosophy)
mesenchymal stem cells, hypoxia, ascorbate, glucose, aging, regenerative medicine, reprogramming
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